Rapid resistance detection of resistance level is most important step to manage the resistant population in Tetranychus urticae in Korea. Residual contact vial bioassay (RCV) and quantitative sequencing (QS) methods were employed to determine the resistance level and applied to measure the resistance level of 46 field population collected from rose, strawberry and apple trees. Most populations exhibited multiple resistance pattern to various types of acaricides. Especially, the resistance levels in mites from rose cultivation area were higher than those from strawberry and apple cultivation areas. The resistance level detection would provide a useful parameter enabling the proactive action for a proper resistant population management for T. urticae.

The two-spotted spider mite, Tetranychus urticae, is one of the most important pests of crops. However, chemical acaricides are not always effective and their continuous use has resulted in resistance among two-spotted spider mite populations. To reduce or replace use of chemical acaricides, the entomopathogenic fungi having acaricidal activity were investigated. As results, 12 isolates could be selected from 342 isolates of fungi against two-spotted spider mite. To show the multiple roles of entomopathogenic fungi against plant pathogen, the antimicrobial activity of selected 12 isolates were evaluated to plant pathogenic bacterium Ralstonia solanacearum and fungus Botrytis cinerea. Various antimicrobial activities were observed against these plant pathogens. Consequently, these entomopathogenic fungi would be offered an alternative for dual control agents against the two-spotted spider mite and plant diseases.

Recently, RNA interference (RNAi) technology has been emerged as a potent tool for pest control strategy. Based on the previous studies on RNAi via leaf disc-mediated systemic delivery of dsRNA and in planta expression of hairpin RNA by agroinfiltration, the coatomer subunit alpha (COPA) gene has been found to be a crucial target for RNAi against Tetranychus urticae. In current study, transgenic plants of Arabidopsis thaliana expressing COPA hairpin RNA were generated by the floral dip method. Putative transgenic plants were screened by PCR and positive transformants were subjected to bioassay using age-synchronized and host-adapted T. urticae. T. urticae feeding on plants expressing dsRNA/siRNA showed more than 80% mortality as compared to the mites feeding on control plants at 6 days post-infestation. Our data shows that in planta expression of hairpin gene such as COPA may serve as an effective way for the control of this important pest in ornamental and economically important plants.

In our previous study, COPA (coatomer subunit alpha) gene from the two-spotted spider mite, Tetranychus urticae, exhibited RNA interference (RNAi)-based lethality when its double-stranded RNA (dsRNA) was systemically delivered via multi-unit chambers or its hairpin RNA was in planta-expressed by agroinfiltration. The cumulative mortality of T. urtcae was 55.0 ±14.2% in soybean plants agroinfiltrated with COPA gene. To investigate the temporal expression profiles of hairpin RNA following agroinfiltration, the amount of hairpin RNA expressed in plants was quantified over time by quantitative real-time PCR. Relative transient expression levels of T. urticae COPA hairpin RNA was highest at 46 h post-agroinfiltration and the extent of COPA gene knockdown was lowest at 12 h post-infestation on soybean plants. To investigate small interference RNA (siRNA) profiling, northern blot assay is currently under progress.

국내 시설포도 재배면적은 2006년 1,824ha에서 2012년 2,591ha로 42.1% 증가되었다(통계청). 이후에도 수입과실에 대한 경쟁력 강화를 위해 지속적으로 늘어 응애류, 총채벌레류, 애매미충류, 깍지벌레류 등의 미소해충 발생이 증가되고 있다. 이 중 잎응애인 점박이응애(Tetranychus urticae), 차응애(T. kanzawai), 사과응애(Panonychus ulmi)가 포도원에서 발견되었고, 점박이응애가 우점종으로 알려져 있다. 시설재배시 고온으로 인해 해충 번식속도가 빠르고 연중 번식이 가능하여 많은 회수의 약제 살포가 필요하며, 이로 인해 농가의 방제비용 및 노동력이 많이 들어 어려움을 호소하고 있다. 본 연구는 초미립 자동연무기(삼영바이오, NSY-350)를 이용하여 시설포도원에서 점박이응애의 방제효율을 검정하고자 수행하였다. 10a 기준 관행 동력분무기에 비해 80.3%의 약제량과 3.6%의 물량으로 사이에노피라펜 액상수화제를 살포했을 때 점박이응애를 100% 방제하였다. 살포준비를 위한 시간도 관행에 비해 10%밖에 안 들었으며, 포도잎이 겹친 사이에 농약 흡착지를 놓고 농약부착능력을 검정한 결과, 관행 일반분무기에 비해 부착효과가 인정되었다.

The two spotted mite, Tetranychus urticae Koch, is a global pest, and has developed severe resistance to several types of acaricides. This study compared the development in susceptible (S) and acequinocyl, bifenazate, pyridaben, abamectin and etoxazole-resistant (AcR, BR, PR, AbR and ER) strains of T. urticae by X-ray irradiation. When eggs were irradiated with 100 Gy, the egg hatching was completely inhibited in all strains. When nymphs were irradiated with 200 Gy, their hatching was completely inhibited in all strains. When adults were irradiated, the fecundity and egg hatching was decreased at 150 Gy and above, and hatchability of F1 generation was completely inhibited at 300 Gy in all strains. Adult longevity was slightly increase at 150 Gy and above. In addition, we performed quantitative real-time PCR on several genes. To examine the difference of all strains on radiation stress-induced gene expression, we performed quantitative RT-PCR (qPCR) of several known stress-induced genes.

RNA interference (RNAi) has been proven as an operative technique for efficient gene silencing in many organisms. In our study, Tetranychus urticae, an extremely polyphagous and rapidly resistance developing mite against acaricides, was screened by double-stranded RNA (dsRNA) delivery method using multi-unit chambers. Among several lethal genes of T.urticae, COPA (the coatomer subunit alpha), a gene involved in membrane transport between the endoplasmic reticulum (ER) and the Golgi complex, showed the highest mortality rate [median lethal time (LT50)=54h]. To investigate the effect of dsCOPA treatment to lysosome formation, we used the Lysotracker green DND26 dye, selective to acidic cellular compartments such as lysosome. The result revealed that the dsEGFP-treated T. urticae has 1.3-fold more of lysosome than that of dsCOPA treated, indicating that downregulation of COPA affected lysosomes function and autophagy, thereby resulting in lethality. To investigate the further detailed toxic mechanism of COPA knockdown, investigation on histological changes in T. urticae fed COPA dsRNA is currently on going.

본 실험은 유기농 신선초(Angelica utilis)포장에서 점박이응애(Tetranychus urticae)의 발생지점 및 확산 양상을 확인하고자 수행하였다. 실험은 17주 동안 강원도 원주시 신림면 용암리에 위치한 유기작목반 비닐온실(625㎡/동)에서 진행되었다. 온실 입구부터 5m 간격으로 확대경을 이용해 해충 밀도를 조사한 결과 A농가에서는 1주차에 입구에서 95m 지점의 해충밀도가 가장 높았으며, 시간이 경과함에 따라 앞쪽으로 확산되었다. 이는 A농가 뒤쪽 우거진 수풀에서 점박이응애가 유입되어 도로변이 있는 앞쪽으로 확산된 것으로 보인다. B농가의 경우 1주차에 입구에서 50m 지점의 해충밀도가 가장 높았으며 시간이 경과함에 따라 뒤쪽으로 확산되었다. 이는 B농가 앞쪽에 있는 신선초온실에서 해충이 유입되어 확산된 것으로 보인다. 또한 점박이응애의 천적을 1주일간격으로 3회 방사한 결과 A농가는 방사 6주차에, B농가는 방사 3주차에 100% 해충방제효과를 보였다. 신선초는 1년 상시 재배되는 작형인 만큼 계절별, 지리적 특성에 따라 해충 양상이 달라질 수 있으므로 추후 체계적인 모니터링을 통해 효과적인 천적방사시기 및 방사지점을 확인한다면 해충발생초기에 소량의 천적투 입으로 방제효과 극대화, 노동력과 방제비용의 절감을 기대할 수 있을 것으로 기대된다.

Tetranychus urticae is extremely hard to control by conventional acaricides due to its rapid development of resistance to nearly all arrays of acaricide. As an alternative control measure of acaricide-resistant mites, RNA interference (RNAi)-based method has recently been suggested. A double-stranded RNA (dsRNA) delivery method using multi-unit chambers was established and employed to screen the RNAi toxicity of 42 T. urticae genes. Among them, the dsRNA treatment of coatomer I (COPI) genes, such as coatomer subunit epsilon (COPE) and beta 2 (COPB2), resulted in high mortality [median lethal time (LT50) = 89.7 and 120.3 h, respectively]. The transcript level of the COPE gene was significantly (F3,9 = 16.2, P = 0.001) reduced up to 24% following dsRNA treatment, suggesting that the toxicity was likely mediated by the RNAi of the target gene. To identify the deferentially expressed gene upon dsRNA ingestion, RNA-seq was employed to compare the transcriptional profiles between mites fed dsEGFP and dsCOPB2. Approximately 928 of genes were up- or down-regulated significantly (P < 0.05) compared to control and 182 genes were commonly responded to the treatment of both dsRNAs. Those dsRNA-responsible genes were mainly categorized into metabolic enzymes, transporters and secretory proteins. Further study would be necessary to elucidate the roles of dsRNA-responsible genes in mite’s dsRNA uptake and defense.

Expression of hairpin RNA corresponding to the part of COPA transcript was done by agroinfiltration in soybean plants and was confirmed by qRT-PCR. In a pot experiment, T. urticae was infested on agroinfiltrated soybean plants and T. urticae mortality was observed and compared with control plants overtime. Significantly higher mortalities of T. urticae were observed in the COPA-agroinfiltrated soybean plants from post-infestation day 2 (15 ±5%), day 4 (50 ±10 %). At post-infestation day 6, mortality reached to (70 ± 15%). To validate the observed COPA silencing effect in T. urticae fed on the agroinfiltrated soybean plant expressing COPA hairpin RNAs, qRT-PCR analysis was performed. The transcript level of COPA gene was decreased in T. urticae fed on agroinfiltrated soybean plants expressing COPA hairpin RNA from post-infestation day 2. At post-infestation day 2, 4 and 6, COPA transcript levels were reduced by 23.8, 20.7 and 18.8 fold, respectively compared to post-infestation day 1 (control). The results obtained in this study also ruled that the plant mediated production and uptake of silencing (dsRNAs/siRNAs) is an effective way to trigger RNAi in the T. urticae.

사과 재배포장에서 점박이응애(Tetranychus urticae Koch)의 월동은 사과나무의 여러 장소에서 5~15마리의 성충 암컷이 개체군을 형성하 여 월동하는 것으로 밝혀졌다. 월동중인 암컷들은 대부분 복숭아순나방(Grapholita molesta) 유충의 고치가 있는 조피사이의 갈라진 틈에서도 발 견되었으며, 부주지 상에 형성된 좁은 조피틈과 과실 수확 후 남아있는 꽃자루에서도 발견되었으나 월동 개체수는 매우 적었다. 또한, 월동중인 암컷들은 결과지의 기부에 형성된 거친껍질 및 눈의 기부, 여러 작은가지의 분지부위 좁은틈에서도 발견되었다. 한편, 수체상에 형성된 일부 점박 이응애 월동장소로 죽은 개각충속, 깍지벌레 빈껍질속, 진딧물의 미라속에서도 발견되었는데, 이들 장소에서의 월동 응애수는 매우 적었다. 2년 에 걸쳐 수행된 본 연구에서 점박이응애의 월동중 암컷 성충의 사망률은 평균 72~80%로 매우 높았으며, 이는 이들 응애가 겨울 동안 추위에 견 디는 저항력이 낮기 때문으로 판단되었다.

점박이응애(Tetranychus urticae)는 시설작물 및 과수류의 주요 해충중 하나로 다양한 방법으로 방제를 수행하고 있고, 그 중 살비제를 이용한 화학적방제가 많이 사용이 되고 있다. 본 연구에서는 우선 배과원에서 화학적 방제에 이용되고 있는 살비제들의 살비효과를 검토하였다. 이를 위하여 충남지역의 공주, 논산, 예산의 각 지역 배 과원 2곳씩을 선정하여 점박이응애를 채집한 후 계통별로 선발된 약제를 처리를 하였다. 세 지역에서 모두 방제가가 높은 것은 Cyflumetofen, Chlorfenapyr, Bifenazate로 72시간 경과 후 90%이상의 높은 방제가를 보였다. 그에 반해 Bifenthrin, Spirodiclofen, Etoxazole, Fenpyroximate, Fenazaquin은 지역마다 방제가가 차이가 있었다. Spirodiclofen의 경우 공주B과원은 77%의 사충율을 보인반면 논산A과원에서는 15%로 두 과원에서 큰 차이를 보였고, Fenpyroximate의 경우에는 공주B과원은 48%의 사충율을 보인 반면 논산A과원 에서는 80%의 높은 사충율을 나타냈다. 이 결과로 보았을 때 지역별로 서로 다른 계통의 약제에 대해 저항성이 발달한 것으로 사료되어 추후에 저항성 발달정도를 알아보기 위한 실험을 진행할 예정이다.

Due to its rapid development of resistance to nearly all arrays of acaricide, Tetranychus urticae is extremely hard to control using conventional acaricides. As an alternative control measure of acaricide-resistant mites, RNA interference (RNAi)-based methods have recently been suggested. A double-stranded RNA (dsRNA) delivery method using multi-unit chambers was established and employed to screen the RNAi toxicity of 42 T. urticae genes. Among them, the dsRNA treatment of coatomer I (COPI) genes, such as coatomer subunit epsilon (COPE) and beta 2 (COPB2), resulted in high mortality [median lethal time (LT50) = 89.7 and 120.3h, respectively]. The transcript level of the COPE gene was significantly (F3,9 = 16.2, P =0.001) reduced by up to 24% following dsRNA treatment, suggesting that the toxicity was likely mediated by the RNAi of the target gene. As a toxicity enhancement strategy, the recombinant dsRNA was generated by reciprocally recombining half-divided fragments of COPE and COPB2. The two recombinant dsRNAs exhibited higher toxicity than the respective single dsRNA treatments as determined using LT50 values (79.2 and 81.5h, respectively). This finding indicates that the recombination of different genes can enhance RNAi toxicity and be utilized to generate synthetic dsRNA with improved RNAi efficacy.

Tetranychus urticae was collected from greenhouse roses to monitor the development of acaricide resistance. Dose-mortality lines were estimated on 16 regional populations with 13 acaricides. For each acaricide, LC50s of the populations were plotted to check normality. LC50s of eight acaricides showed normal distribution and five others did not. An index of Ln (recommended dose/LC50 for each acaricide) checked the development of resistance to populations. The index is based upon recommended dose to control a pest stage and empirical LC50 got from serial dilution range for each pesticide. We tried to categorize acaricides by index due levels of effectiveness to mites: tebufenpyrad, fenpyroximate, bifenthrin, and fenbutatin oxide as non-effective acaricides with less than index 1.0, chlorpyrifor+bifenthrin and milbemectin as alert level placed between 1.0 and 2.0, acequincyl as caution between 2.0 and 3.0, and abamectin, cyflumetofen, bifenazate, chlorfenapyr+fulacrypyrim and propargite as effective over 3.0. We also tested the similarity of acaricide actions for choosing effective acaricides against resistant mites and of populations for resistance management. We could make several acaricides groups: group1 including abamectin, cyflumetofen, and bifenazate group 2 propargite and chlorpyrifos+bifenthrin group 3 chlorfenapyr and acequinocy and group 4 mibemectin, fenyroximate, and bifenthrin, by which we can suggest not to use acaricide within the same group to avoid the resistance development. Populations grouping would imply similar practices of acaricide use, so we can manage pesticide usage, effectively. Group A includes Gimhae 2, 3, 4, 5, Jincheon2, and Taean, and Group B includes Goyang, Gangjin1, Paju, Gangin2 and Namwon.

The two-spotted spider (Tetranychus urticae) is one of the most serious pests worldwide and has developed resistance to almost all types of acaricides. Various mutations on acaricide target and detoxification genes and their duplication (including overexpression) have been identified since the completion of T. urticae genome analysis. The mutations are mainly observed in functionally important domains (i.e. transmembrane, cellular loops and catalytic triad, etc.), which likely confer acaricide resistance directly or indirectly. Gene duplication was found on major detoxification and insecticide target enzymes, such as cytochrome P450, glutathione S-transferase, ABC-transporter, UDP-glycosyltranferase and acetylcholinesterase (AChE). Interestingly, co-occurrence of both mutation and gene duplication (especially, single gene amplification) was found in AChE, which possibly explains the compensatory role of gene duplication to minimize the fitness cost mediated by point mutations. Such mutation and duplication traits associated with resistance can be utilized as molecular markers for the determination of resistance levels based on the quantitative sequencing and real-time PCR.

This study were performed in Tropical Plant Resources Research Center of Korea National Arboretum, Pocheon, Korea to assess the effects of beneficial insects against Pseudococcus comstocki (Kuwana) on Phyllanthus acidus (Linnaeus) Skeels and Tetranychus urticae Koch on Acalypha wilkesiana Muell Arg. We investigated the density of leaf-feeding P. comstocki at intervals of approximately 2 weeks and T. urticae at intervals of approximately 1 weeks after introducing their natural enemies; Chrysoperla nipponensis (Okamoto) and Phytoseiulus persimilis Athias-Henriot. The densities of P. comstocki were suppressed 80.0% (after 4 weeks) and 97.5% (after 8 weeks) after introducing C. nipponensis. The densities of T. urticae were suppressed 84.1% (after 4 weeks) and 99.6% (after 6 weeks) after introducing P. persimilis.

이 연구는 사과원에서 20년동안 두 종 응애의 밀도변화를 조사하기 위하여 수행하였다. 사과나무를 가해하는 두 종의 응애 해충, 점박이응애 (Tetranychus urticae)와 사과응애(Panonychus ulmi)의 발생을 1992년부터 2011년까지 8개 도시 포함, 한국의 남부지역에 위치한 사과주산지역에 서 조사하였다. 20년간의 추세는 많은 과원이 1992년부터 1999년까지는 점박이응애가 주로 가해를 하였지만 2000년도부터는 사과응애가 우점하 기 시작했다. 사과응애의 평균관찰밀도는 일정했고 점박이응애의 밀도는 이 기간동안 일정하지 않았다. 5년주기별 발생추세 분석은 점박이응애의 밀도가 2002년 이후에 감소하는 것을 보여주었다. 4월부터 9월까지 월별피해과원율과 두종 응애의 평균발생밀도는 사과응애의 밀도가 4월에 높지 만 점박이응애의 밀도가 5월부터 9월까지 높았다. 이런 변화는 살충제 살포횟수, 초경재배관리, 질소비료 사용의 감소와 전체적인 과원관리의 변화 등으로 기인한다. 그러나 이러한 예측은 이를 증명하기 위해 보다 자세한 연구가 필요하다. 본 연구를 통해 비료살표와 같은 재배법과 살충제 살포횟 수와 종합적해충관리 등의 환경적 변화가 사과원에서 두종 응애의 우점종과 발생밀도에 영향을 준 것으로 보인다.

This study compared the development, reproduction, and DNA damage in insecticide (acequinocyl, bifenazate, and etoxazole)-resistant (AR, BR, and ER) and -susceptible (S) strains of Tetranychus urticae by electron beam irradiation. When eggs were irradiated with 150 Gy, the egg hatching was completely inhibited in all strains. When nymphs were irradiated, their emergence rate was decreased with increasing doses of electron beam irradiation. When adults were irradiated, the longevity and the number of eggs was not affected at all target dose in all strains. However, hatchability of F1 generation was perfectly inhibited at 400 Gy in all strains. The DNA damage caused by electron beam irradiation was evaluated by an alkaline comet assay. These results suggest that electron beam irradiation induced abnormal development and reproduction in S, AR, BR, and ER strains of T. urticae.

The two spotted mite, Tetranychus urticae Koch, is a global pest, and has developed severe resistance to several types of acaricides. Acequinocyl-resistant strain and bifenazate-resistant strain of T. urticae showed higher relative activity level of glutathion-S-transferases (GSTs) than susceptible strain. Studies of resistance in T. urticae have focused on classical detoxifying enzyme system, such as GSTs. We examined the expression of six GSTs genes in T. urticae belong to five different cytosolic classes, including two in delta, one in omega, one in mu, one in zeta, and one mitochondrial GSTs class, kappa. RT-qPCR results showed that, among these six GSTs genes, the relative expression level of delta 1 was significantly higher in bifenazate-resistant strain than in other strains (susceptible, abamectin, acequinocyl, etoxazole and pyridaben strain). Expression level of the delta 2 was also higher in acequinocyl-resistant strain than in other strains. The elevated GSTs gene transcripts following exposure to acequinocyl, bifenazate might be one of the mechanisms involved in detoxification of these acaricide. These results may be caused by different mechanisms in the resistance development of T. urticae to these two different types of acaricides.